Gallstone disease is one of the most common and costly digestive diseases. Female sex hormones have been implicated as contributory factors to the promotion of gallstones. Female sex hormones promote gallstone formation by increasing biliary cholesterol saturation and gallbladder motility. Hepatic secretion of cholesterol saturated bile is a necessary, but not sufficient, prerequisite for gallstone formation, since cholesterol-supersaturated bile is common in healthy population suggesting that additional defects are required for gallstones to occur. It has been established that nucleation defect exists in the patients with gallstones although the exact mechanism of the abnormality is unknown. Anti- and pro-nucleating agents have been identified and play critical role in gallstone formation. Nucleation of cholesterol crystal in bile precedes from cholesterol rich phospholipid vesicles. Gallstone formation also requires pathologic changes within the gallbladder. Several studies in male animal models have shown that gallbladder Na+ and H2O absorption increase prior to gallstone formation and promote cholesterol nucleation. Na+/H+ exchange (NHE) is a major pathway for gallbladder Na+ transport and is upregulated prior to crystal formation in male prairie dogs. Recent studies suggest that estrogens influence both cholesterol nucleation in bile and regulate epithelia NHE. Estrogen receptors (ER) have been documented in gallbladders of gallstone patients and the proportion of gallbladders expressing ER is higher in women than in men. This proposal seeks to synergistically utilize innovative, exploratory and complementary expertise in the gallbladder epithelial ion transport and in chemical engineering as it relates to gallstone formation. We propose to test two separate hypotheses where gender differences may exist to explain the increased incidence of gallstones in women. Hypothesis I will test that: 1) Cholesterol nanodomains are readily formed in bile from women, thereby causing a nucleation defect and making them susceptible to develop gallstones; and 2) sex hormones play a key role in these processes. Hypothesis II will test that: 1) Females are predisposed to increased gallbladder Na+ and water absorption; and 2) sex hormones play an important role in regulating gallbladder NHE stimulating Na+ absorption and promoting gallstone formation. These studies will be performed in both male and female prairie dog gallstone model using physicochemical, biochemical, immunochemical and molecular biological techniques. The results from these studies will enhance our understanding of the gender differences in cholesterol nucleation and gallbladder Na+ transport and help identify molecular targets that can be exploited to develop novel therapies for the prevention and treatment of gallstone disease in women.